Abstract Replicative aging of budding yeast, Saccharomyces cerevisiae, has been a remarkably useful model for aging studies, providing fundamental genetic and molecular insights into aging. Studies of chromatin biology have also benefited from the yeast model, since many molecular mechanisms of chromatin are highly conserved among eukaryotes. The goal of this proposal is to establish two screening approaches that are built on previous works of mine and others for a better understanding on how epigenetics and chromatin regulatory pathways are involved in the process of aging. The results of these screenings are expected to identify many new targets and chromatin regulators for aging studies. Epigenetic changes, including histone post-translational modifications, are critical regulatory mechanisms, involved in all developmental processes including aging. However, most studies, and our previous research in this area have focused on only a few known histone modification targets of Sirtuins. It is of tremendous interest to discover new histone targets of Sirtuins and other chromatin-mediated aging regulators. I propose to utilize an existing systematic histone mutant library to establish a novel screening approach based on the well-established old cell sorting method, and to screen for histone mutants that alter replicative lifespan of yeast (aim 1). The available histone mutant library was developed by Jef Boeke's group. It contains systematic mutations for histone H3 and H4, with multiple versions of mutations for each residue. More importantly this library features mutations in an integrated form, which is a critical factor for use in aging studies. The cell surface labeling and sorting method was development by Leonard Guarente's lab and was used in my previous work to isolate old yeast cell in 108 quantities. The proposed novel screening approach builds on this method and enriches for long-lived cells in the old cell fraction from a pool of histone mutant strains. This approach allows high throughput screening for mutations that extend or shorten lifespan without performing the tedious lifespan assay for all mutant strains. Hits from the screening will be validated by conventional lifespan assay. The yeast deletion library, a collection of yeast strains, each harboring a single gene deletion, has been a tremendously valuable resource for defining gene function, understanding the structure and logic of multiple cellular pathways, many of which are conserved from yeast to humans, and for identifying the target and mode of action for several promising drugs. This collection was recently harnessed by Kennedy and Kaeberlein's groups to identify novel aging regulators and pathways. However, it has become exceedingly difficult to explorer more pro-lifespan regulators, like Sir2, because disruption of many genes that impair critical pathways naturally result in shorter lifespan and they may not be directly involved in aging regulation. Those long-lived deletion mutants are often confusing because many are involved in important biological processes. Therefore evaluate aging and biological effects of over-expressing each gene and compare to the results from the deletion library become more relevant and efficient in identifying genes that promote longevity, like Sirtuins. I propose to construct a collection of yeast strains allowing for systematic over-expression of yeast genes using high-throughput methods, and then to use this collection to discover novel mechanisms underpinning the biology of aging and age-related diseases (aim 2). I will take advantage of a readily available yeast gene plasmid collection FLEXgene, which contains more than 5,500 yeast genes, and the highly-efficient gateway cloning system to generate a library of yeast plasmids with an appropriate marker for integration and selection. Most of the work, including plasmid preparation, gateway cloning, yeast transformation and verification will be performed with a state-of-the-art liquid handling robot recently acquired by my mentor. An initial screen of 400 chromatin related genes is proposed to complete in specific aim 2 during this project timeframe. I expect to identify novel aging regulators that are either directly involved in chromatin biology or have direct targets in chromatin.